Figure 2. Rubicon and Pacer RH domain have inverted pRAB7A binding specificity [FINAL]

Figure 2. Rubicon and Pacer RH domain have inverted pRAB7A binding specificity. (A) Domain maps of Rubicon and Pacer. Rubicon and Pacer both bind UVRAG via their PIK Binding Domains (PIKBD), and both possess Rubicon Homology (RH). (B) PhosTag gel showing in vitro phosphorylation of RAB7A using recombinantly purified TBK1. Here, TBK1 was mixed with Q67L, GTP-locked RAB7A for 3h at room temperature, supplemented with ATP and Mg. PhosTag gels slow the migration of phosphorylated proteins. (C) Confocal based fluorescent bead-binding assay schematic. Amylose beads were functionalized with MBP-RH domain, and then incubated in solutions of either phosphorylated or unphosphorylated RAB7A chemically conjugated to a fluorescent dye. (D) Amylose beads were incubated in 500 nM MBP- RH domain and 2 μM Alexa Fluor 647647-labelled RAB7A prior to washing and imaging on a confocal setting. (E) Crystal structure of the Rubicon RH domain in complex with RAB7A (top, PDB: 6wcw), and a homology model of the Pacer RH domain in complex with RAB7A (bottom, SwissModel). Coloration represents electrostatic potential (blue = positive charge, red = negative charge).(F) Isothermal Titration Calorimetry (ITC) injection plot (left) and binding curve (right) of Rubicon binding to RAB7A. 13 injections performed at 25 C with 150 second delays between injection. Data was fit to a binding curve assuming a simple, single binding site mechanism. Binding curve best fit suggest n = 0.7.